Aquatic Toxicology 160 (2015) 69–75
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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Dose-dependent compensation responses of the
hypothalamic-pituitary-gonadal-liver axis of zebrafish exposed to the
fungicide prochloraz
Yao Dang a , John P. Giesy b,c,d,e , Jianghua Wang a,∗ , Chunsheng Liu a,∗
a
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada
c
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
d
School of Biological Sciences, University of Hong Kong, SAR, Hong Kong, China
e
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China
b
a r t i c l e
i n f o
Article history:
Received 8 November 2014
Received in revised form 5 January 2015
Accepted 7 January 2015
Available online 8 January 2015
Keywords:
HPG axis
Compensation responses
Zebrafish
Prochloraz
Aromatase inhibitor
a b s t r a c t
Compensation responses and adaptability of hypothalamic-pituitary-gonadal (HPG) axis have been
reported in fish exposed to model chemicals, however due to its importance in predictive toxicology
further study was needed to elucidate details of the integrated responses to model chemicals. Transcriptional profiles of the hypothalamic-pituitary-gonadal (HPG) axis and concentrations of 17␤-estradiol (E2)
in plasma were measured in male and female zebrafish that had been exposed to one of seven concentrations of the fungicide, prochloraz: low (1, 3 or 10 ␮g/L), medium (30 or 100 ␮g/L) or high concentrations
(300 or 1000 ␮g/L) for 4 days. In zebrafish exposed to the low and medium concentrations of prochloraz,
compensation responses of the HPG axis through transcription, occurred in brain (up-regulation of gnrh,
gnrhr and lhˇ) and both brain and gonad (up-regulation of steroidogenic genes), respectively. Concentrations of E2 in plasma and expression of estrogen receptor 1 (er1) and vitellogenins (vtgs) in liver did not
change. This result suggested that compensatory responses were successful in maintaining homeostasis. In zebrafish exposed to the two greatest concentrations, compensatory responses occurred in brain,
gonad and liver through up-regulation of er2ˇ, but it failed to maintain concentration of E2 in blood
plasma and expression of er1 and vtgs in liver. Collectedly, the results observed in this study allowed
characterization of dose-dependent compensatory responses along the HPG axis and liver and identified key linkages between compensatory responses occurring in brain, gonad and liver after exposure to
prochloraz.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
As an imidazole fungicide, prochloraz is registered for various
agricultural uses throughout the world (http://pubchem.ncbi.
nlm.nih.gov/summary/summary.cgi?cid=73665). This chemical
can act as an inhibitor of cytochrome P450 (CYP) 14␣-demethylase
(CYP51), which is a key enzyme in the synthesis of ergosterol, and
therefore inhibits growth of fungi (van den Bossche et al., 1987,
1982). However, prochloraz can also inhibit activities of other CYP
enzymes, including cytochrome P450 c17␣-hydroxylase/17,20lyase (CYP17) and aromatase (CYP19) (Ankley et al., 2009). In
∗ Corresponding authors. Tel.: +86 27 87282113; fax: +86 27 87282114.
E-mail addresses: whtjwjh@163.com (J. Wang), liuchunshengidid@126.com
(C. Liu).
http://dx.doi.org/10.1016/j.aquatox.2015.01.003
0166-445X/© 2015 Elsevier B.V. All rights reserved.
vertebrates, the gene product of CYP17 is responsible for synthesis of testosterone (T) and CYP19 catalyzes conversion of T
to 17␤-estradiol (E2). Therefore, inhibition of activities of the
two enzymes would decrease production of both T and E2. These
effects have been previously confirmed experimentally, both
in vitro and in vivo, in mammals, where exposure to prochloraz
significantly inhibited activities of both CYP17 and CYP19 enzymes
and decreased concentrations of T and E2 in blood plasma
(Blystone et al., 2007; Mason et al., 1987; Noriega et al., 2005;
Sanderson et al., 2002; Vinggaard et al., 2000). In fish, treatment
with prochloraz decreased concentrations of T and E2 in blood
plasma, and affected reproductive function (Ankley et al., 2005,
2009; Marca Pereira et al., 2011a, b; Liu et al., 2011; Skolness et al.,
2011; Villeneuve et al., 2007; Zhang et al., 2008a,). Due to its effectiveness as an inhibitor of CYP17 and CYP19 enzymes, prochloraz
has been used as a model chemical for studying the responses of
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Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
hypothalamic-pituitary-gonadal (HPG) axes of vertebrates (Ankley
et al., 2005, 2009; Marca Pereira et al., 2011a, b; Liu et al., 2011;
Villeneuve et al., 2007; Zhang et al., 2008a).
The HPG axis is a dynamic endocrine system, that maintains
physiological conditions of reproduction by various homeostatic
feedback mechanisms during exposure to stressors including
chemicals (Ankley et al., 2009). Compensatory responses of the
HPG axis have been documented in fish exposed to model chemicals (Ankley et al., 2009; Liu et al., 2011; Skolness et al., 2011;
Villeneuve et al., 2007; Zhang et al., 2008a, b, c). For example,
results of several studies have demonstrated that exposure to
fadrazol, which inhibits the enzymatic activity of CYP19, resulted
in decreased concentrations of E2 in blood plasma and caused a
time- and dose-dependent up-regulation of genes of HPG axis in
small fish. Up-regulation of those genes was considered to be a
compensatory mechanism to return concentrations of E2 in blood
plasma to pre-exposure values (Villeneuve et al., 2009; Zhang
et al., 2008c). Similarly, treatment with prochloraz significantly
decreased concentrations of T and E2 in blood plasma and resulted
in a comprehensive up-regulation of genes along the HPG axes
of fishes (Ankley et al., 2009; Liu et al., 2011; Skolness et al.,
2011; Zhang et al., 2008a). Furthermore, in two time-course studies, inhibitory effects of prochloraz or fadrozole on production of
E2 in fathead minnow were transitory and did not persist during
the 8-day exposure phase, which demonstrated the effectiveness
of compensatory responses. Termination of the exposure resulted
in recovery of expression of genes and concentrations of steroid
hormones, including a brief period of “overcompensation” immediately after cessation of exposure (Ankley et al., 2009; Villeneuve
et al., 2009). These results suggest key compensatory responses of
the HPG axis after exposure to chemical stressors, and highlight
the need to consider these compensatory responses when developing approaches to assess potential risks of chemicals (Ankley et al.,
2009; Villeneuve et al., 2009).
To better understand compensatory mechanisms in fishes and
more accurately predict effects of chemicals based on modes
of action a more comprehensive characterization of these dosedependent compensatory responses was still needed. Objectives of
the present study were to: (1) examine dose-dependent expression
behaviors of HPG axis and genes expressed in liver; (2) compare
sensitivities of genes; and (3) identify linkages between responses
occurring at different organs, including brain, gonad and liver of
zebrafish.
2. Materials and methods
2.1. Chemicals and reagents
Prochloraz and TRIzol regent were obtained from Sigma (St.
Louis, MO, USA) and Invitrogen (New Jersey, NJ, USA), respectively. Reverse transcription and SYBR Green kits were purchased
from Takara (Dalian, Liaoning, China). 17␤-estrogen (E2) enzyme
immunoassay (EIA) kits were obtained from Cayman Chemical
Company (Ann Arbor, MI, USA). All the other reagents used in this
study were of analytical grade.
2.2. Fish and chemical exposure
Zebrafish were maintained in flow-through tanks at 28 ± 0.5 ◦ C
with a 12:12 light/dark cycle, and water pH, hardness and dissolved
oxygen were routinely monitored. Before exposure, 5-month old
males and females (sexual maturity) were acclimated in 15-L tanks
filled with 10 L of carbon-filtered water for 1 week. After acclimation, fish were exposed to 0, 1, 3, 10, 30, 100, 300, 1000, 3000
or 10,000 ␮g/L (0, 0.0027, 0.0080, 0.027, 0.080, 0.27, 0.80, 2.7, 8.0
or 27 ␮M) prochloraz for 4 days. Concentrations were selected
based on previous studies, where comprehensive compensation
responses of HPG axis genes would be expected to occur (Ankley
et al., 2009; Liu et al., 2011; Zhang et al., 2008a). Five females and
five males were exposed in each of 2 replicated tanks for each
concentration. One half of the water in each tank was replaced
daily with fresh carbon-filtered water containing corresponding
concentration of prochloraz. Both control and exposure groups
received 0.01% DMSO since previous study demonstrated that such
DMSO concentration did not affect reproductive function (Han
et al., 2013). During the exposure period, survival was recorded.
After exposure, fish were euthanized and blood was collected for
plasma hormone analysis as described before (Liu et al., 2009).
Tissues from brain (including hypothalamus and pituitary), gonad
and liver were sampled and preserved in TRIzol reagent for subsequent RNA isolation. In this study, experimental procedures were
carried out following the approved protocol by Institutional Animal Care and Use Committee (IACUC) of Huazhong Agricultural
University.
2.3. Quantification of hormones
Briefly, plasma was obtained by centrifugation (5000 × g for
5 min at 4 ◦ C) of whole blood. Plasma from 2 fish was pooled for
quantification by use of a commercial EIA kit as described previously (Liu et al., 2009). Briefly, plasma (3 ␮L) from each pooled
sample was diluted with 500 ␮L ultrapure water and extracted
thrice with 2 mL of ethyl ether, and the ether phase was collected
and evaporated. After that, residues were redissolved with EIA
buffer provided in the kit and E2 was quantified following manufacturer’s instructions. The limit of quantification was 6 pg/L, and
the intra- and inter-assay coefficients of variance (CV) were <10%.
Each sample was quantified 3–4 times. In order to determine effects
of prochloraz on gene expression and hormone production, concentrations of E2 in plasma were expressed as fold-change relative to
control.
2.4. Quantitative real-time PCR assay
In this study, quantitative real-time PCR was performed using
minimum information for publication of quantitative real-time PCR
experiment (MIQE) guidelines (Bustin et al., 2009). The tissue of
each sample employed for qRT-PCR was from one animal. The
isolation of total RNA was performed using TRIzol regent following manufacturer’s instructions. Purity of RNA was examined by
measuring 260/280 nm ratios and 1% agarose-formaldehyde gel
electrophoresis with ethidium bromide staining. Concentrations
of RNA were estimated by determining absorbance at 260 nm.
After measurement of concentration of total RNA, all RNA samples were diluted to 100 ng/␮L, and equal volume of RNA (5 ␮L)
was used for cDNA synthesis. First-strand cDNA syntheses and
quantitative real-time PCR (qRT-PCR) were performed using commercial reverse transcription and SYBR Green kits (Takara, Dalian,
Liaoning, China), respectively following manufacturer’s instructions. Sequences of primers were designed using Primer 3 software
(http://bioinfo.ut.ee/primer3-0.4.0/primer3/) (Table S1 in Supporting Information). Primer specificity was checked by NCBI BLAST,
and melting curve was employed to check out purity and specificity of PCR productions in each assay. Selection of housekeeping
gene was performed using previous method (Andersen et al., 2004).
Transcription of three housekeeping genes (18S rRNA, gapdh, ˇaction) were tested, and expression of 18S rRNA kept unchanged
in brain, gonad and liver of female and male fish after prochloraz
exposure, therefore it was used as an internal control gene. Thermal cycling was set at 95 ◦ C for 2 min, followed by 40–45 cycles of
95 ◦ C for 15 s and 60 ◦ C for 1 min. Expression of target genes were
Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
71
calculated using the 2−Ct method, and was expressed as fold
change relative to control. Each sample was replicated and the
mean of 4–6 measurements reported.
2.5. Statistical analyses
Statistical analyses were performed using Kyplot Demo 3.0 software (Tokyo, Japan). Normality of data sets was examined using the
Kolmogorov-Smirnow test. If necessary, data were log-transformed
to approximate normality. Homogeneity of variances was checked
by Levene’s test. Differences of relative gene expressions or hormone production between the control and each exposure were
evaluated by ANOVA followed by Tukey’s multiple range test. The
level of significant (Type I error; ␣) for all statistical analyses was
set at P < 0.05. In this study, lowest observed effect concentrations
(LOEC), observed maximal response value (OMRV) and observed
effect concentration points (OECPs) estimates were made using
Dunnett’s Test and the concentration to induce half of the maximal response (EC50 ) estimates were calculated using non-linear
regression analyses (Zhou et al., 2006). Median lethal concentrations (LC50 ) for survival were calculated using linear regression
analyses (SPSS 13.0, Chicago, IL, USA).
3. Results
3.1. Survival
Exposure to prochloraz caused a dose-dependent effect on survival of both females and males (Fig. 1). No significant effects on
survival were observed in female and male fish exposed to 1, 3,
10, 30, 100 or 300 ␮g/L prochloraz for 4 days. However, treatment
with 1000, 3000 or 10,000 ␮g/L prochloraz for 4 days significantly
decreased survival to 90%, 10% and 0% of females, and 40%, 0% and
0% of males, respectively. The calculated median lethal concentrations (LC50 ) for females and males were 2168.3 and 874.4 ␮g/L,
respectively. Therefore, only samples from 0, 1, 3, 10, 30, 100,
300 and 1000 ␮g/L exposure groups were used for hormone measurement and gene expression analysis. The group of fish exposed
to 0 ␮g/L was defined as control group, while 1, 3 and 10 ␮g/L
groups were defined as low concentration exposure groups, 30
and 100 ␮g/L groups were defined as medium concentration exposure groups, and 300 and 1000 ␮g/L groups were defined as high
concentration exposure groups.
Fig. 1. Dose-dependent effect of prochloraz on survival in female and male
zebrafish. Values represent mean ± SEM (n = 2 tanks). Median lethal concentrations
(LC50 ) for females and males were calculated using SPSS (13.0) software (Chicago,
IL, USA). Curves were fitted using the local polynomial regression method.
Fig. 2. Effect of prochloraz on plasma E2 concentration in female and male zebrafish.
Values represent mean ± SEM. Significant differences from the control are indicated
by *P < 0.05 (females) or #P < 0.05 (males). Each concentration contains 3–4 biological replicates, and each replicate contains 2 fish. Data were expressed as fold change
relative to control.
3.2. Production of E2
The average plasma E2 concentrations of females and males
were 833.8 and 377.8 pg/mL in control group, respectively. No
significant effects on concentrations of E2 in blood plasma were
observed in females exposed to 1, 3, 10, 30 or 100 ␮g/L prochloraz, however, exposure to 300 or 1000 ␮g/L prochloraz significantly
decreased concentrations of E2 by −0.57 and −0.67 fold, compared
with the control, respectively (Fig. 2). In males, only exposure to
1000 ␮g/L prochloraz significantly decreased concentrations of E2,
while treatment with lesser concentrations (1, 3, 10, 30, 100 or
300 ␮g/L) did not change concentrations of E2 in blood plasma
(Fig. 2).
3.3. Transcriptional responses in females
Exposure to low concentrations of prochloraz (1, 3 or
10 ␮g/L) resulted in up-regulation of expression of some genes
in brain, while expression of all the genes tested in ovary and
liver were not significantly different from the control (Fig. 3
and S1–S3, see Supporting Information). Genes up-regulated
in brain included gonadotropin-releasing hormone 3 (gnrh3),
gonadotropin-releasing hormone receptor 2 (gnrhr2), gnrhr3,
gnrhr4, luteinizing hormone beta (lhˇ), estrogen receptor 1 (er1)
and er2˛, while the expression of gnrh2, gnrhr1 and fshˇ in brain
kept unchanged compared with the control.
Treatment with medium concentrations of prochloraz (30 or
100 ␮g/L) significantly up-regulated expression of some genes in
brain and ovary, while expression of all genes tested in liver were
unchanged (Fig. 3 and S1–S3, see Supporting Information). Genes
up-regulated in brain after exposure to medium concentrations
of prochloraz, included gnrhr2, gnrhr3, gnrhr4, lhˇ, er1 and er2˛.
Genes up-regulated in ovary after exposure to medium concentrations prochloraz included luteinizing hormone receptor (lhr),
steroidogenic acute regulatory protein (star), cytochrome P450
c17␣-hydroxylase/17,20-lyase (cyp17), 17␤-hydroxysteroid dehydrogenase (17ˇhsd), aromatase (cyp19a) and er2˛.
Expression of some genes measured in brain, ovary and liver
were significantly changed after exposure to high concentrations
of prochloraz (300 or 1000 ␮g/L) (Fig. 3 and S1–S3, see Supporting Information). In brain, expression of gnrhr2, gnrhr3, gnrhr4
and lhˇ was significantly up-regulated after exposure to 300 or
1000 ␮g/L prochloraz, while expression of other genes examined were unchanged compared with those in controls. In ovary,
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Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
Fig. 3. Expression profile of brain, ovary and liver genes in female and male zebrafish exposed to different concentration of prochloraz. Each concentration contains 4–6
biological replicates. Data were expressed as fold change relative to control.
exposure to 300 ␮g/L prochloraz significantly up-regulated expression of fshr, lhr, star, cytochrome P450 side-chain cleavage (cyp11a),
cyp17, 17ˇhsd, cyp19a, er1 and er2˛; exposure to 1000 ␮g/L significantly up-regulated expression of lhr, star, cyp11a, cyp17, cyp19a
and er2˛. In liver, a significant down-regulation in expression of er1,
vitellogenin 1 (vtg1) and vtg2 was observed after exposure to 300 or
1000 ␮g/L prochloraz, while expression of er2˛ was up-regulated
compared with the control.
In order to compare sensitivities and responsive behaviors
between genes tested in brain, ovary and liver of females, LOEC,
EC50 , OMRV and OECPs of each gene were estimated. In general,
based on LOEC and EC50 values, the rank order of gene sensitivities
was genes in brain > in ovary > in liver (Table 1). The rank order of
OECPs were genes in brain > in ovary > in liver (Table 1).
in brain, testis and liver (Fig. 3 and S4–S6, see Supporting Information). In brain, expressions of gnrh2, gnrhr1, gnrhr2, gnrhr3, gnrhr4,
lhˇ, er1 and er2˛ were up-regulated, compared with their levels of
expression control. In testis, expression of star, cyp17 and cyp19a
were significantly up-regulated, while expressions of er1 and er2˛
were down-regulated. In liver, exposure to 1000 ␮g/L prochloraz
down-regulated expression of vtg1, but no statistically significant effects were observed when fish were exposed to 300 ␮g/L.
Treatment with 300 ␮g/L prochoraz significantly up-regulated
expression of er2˛, while exposure to the greater concentration
(1000 ␮g/L) did not change expression of that gene.
Similar to females, the rank order of gene sensitivities in males
was genes in brain > in testis > in liver (Table 1). The rank order of
OECPs were genes in brain > in testis > in liver (Table 1).
3.4. Transcriptional responses in males
4. Discussion
Exposure to low concentrations (1, 3 or 10 ␮g/L) of prochloraz significantly changed expression of some genes in brain, while
expression of all genes measured in testis and liver were not
changed (Fig. 3 and S4–S6, see Supporting Information). Genes
which were changed in brain were gnrh2, gnrhr1, gnrhr2, gnrhr4,
lhˇ, er1 and er2˛.
Treatment with medium concentrations of prochloraz (30 or
100 ␮g/L) caused significant changes in expression of some genes
monitored in brain and testis, while no significant effects on expression of genes monitored in liver were observed (Fig. 3 and S4–S6,
see Supporting Information). In brain, exposure to 30 or 100 ␮g/L
significantly up-regulated expression of gnrh2, gnrhr1, gnrhr2,
gnrhr3, gnrhr4, lhˇ, er1 and er2˛. In testis, expression of cyp17
and cyp19a was up-regulated, while expression of er1 and er2˛
were unchanged. No significant effects on expression of other genes
monitored in testis compared with the control were observed.
Exposure to the greatest concentrations of prochloraz (300 or
1000 ␮g/L) significantly changed expression some genes monitored
Recently, compensatory responses and adaptability of the HPGaxis, represented as up-regulation of genes along this axis, were
reported in fishes exposed to model chemicals, such as fadrozole,
prochloraz, kethconazole and highlighted the need to consider
compensatory responses when developing approaches to assess
potential risks of chemicals (Ankley et al., 2007, 2009, 2012;
Villeneuve et al., 2009a, 2009b, 2013; Breen et al., 2013). For the
first time, the results observed in this study further demonstrated
that in female and male zebrafish exposed to low concentration
prochloraz, transcriptionally compensatory responses of HPG axis
and liver only occurred in brain; in medium concentration groups
the compensatory responses occurred in both brain and gonad; in
high concentration groups, the compensatory responses seemed to
occur in brain, gonad and liver.
Direct effects of prochloraz on concentrations of E2 in blood
plasma and expression of er1 and vtgs in liver of female and male
zebrafish were consistent with prochloraz’s anticipated mode of
action. Prochloraz is an inhibitor of various CYP enzymes. Results of
Table 1
Endpoints determined for gene expression in HPG axis and liver of female and male zebrafish exposed to different concentration of prochloraz.
Organs
Genes
LOEC
EC50
OECP(s)
OMRV
LOEC
EC50
OECP(s)
OMRV
Brain
gnrh2
gnrh3
gnrhr1
gnrhr2
gnrhr3
gnrhr4
fshˇ
lhˇ
er1
er2˛
NE
3
NE
≤1
≤1
≤1
NE
3
≤1
3
NE
2.8 (2.3, 3.2)
NE
0.7 (0.7, 0.7)
0.9 (0.8, 1.0)
0.7 (0.7, 0.7)
NE
23.7 (19.3, 28.1)
0.9 (0.8, 1.0)
2.5 (2.4, 2.6)
NE
3,10
NE
1, 3, 10, 30, 100, 300, 1000
1, 3, 10, 30, 100, 300, 1000
1, 3, 10, 30, 100, 300, 1000
NE
3, 10, 30, 100, 300, 1000
1, 3, 10, 30, 100, 300
3, 10, 30, 100, 300
NE
3.0 (2.2, 3.9)
NE
1.6 (1.5, 1.7)
2.3 (2.2, 2.3)
1.7 (1.6, 1.7)
NE
28.7 (25.2, 32.2)
1.0 (0.9, 1.1)
0.8 (0.7, 0.9)
10
NE
≤1
1
30
≤1
NE
10
≤1
≤1
83.4 (75.5, 91.3)
NE
72.3 (67.0, 77.6)
88.3 (80.4, 96.2)
60.8 (56.4, 65.2)
134.2 (123.7, 144.7)
NE
438.3 (389.2, 487.4)
175.1 (162.0, 188.2)
439.8 (399.5, 480.1)
10, 30, 1000
NE
1, 30, 100, 300, 1000
1, 30, 100, 300, 1000
30, 100, 300, 1000
1, 10, 30, 100, 300, 1000
NE
100, 300, 1000
1, 30, 100, 300, 1000
1, 30, 100, 300, 1000
1.2 (1.2, 1.3)
NE
0.7 (0.7, 0.7)
1.2 (1.1, 1.3)
1.7 (1.5, 1.9)
2.9 (2.5, 3.2)
NE
3.2 (2.7, 3.7)
2.0 (1.8, 2.2)
2.2 (2.0, 2.4)
Gonad
fshr
lhr
hmgr
star
cyp11a
3ˇhsd
cyp17
17ˇhsd
cyp19a
er1
er2˛
300
30
NE
30
300
NE
100
100
30
300
100
NE
31.5 (29.2, 33.8)
NE
NE
98.4 (91.7, 105.7)
NE
105.4 (98.4, 112.4)
130.5 (120.0, 141.0)
125.4 (114.9, 135.9)
NE
85.8 (80.5, 91.1)
300
30, 100, 300, 1000
NE
30, 100, 300, 1000
300, 1000
NE
100, 300, 1000
100, 300
30, 100, 300, 1000
300
100, 300, 1000
2.3 (2.0, 2.6)
3.0 (2.8, 3.2)
NE
4.3 (3.6, 4.9)
1.0 (1.0, 1.0)
NE
2.0 (1.7, 2.3)
2.1 (1.8, 2.3)
7.6 (7.3, 7.9)
1.8 (1.7, 2.0)
0.9 (0.8, 1.0)
NE
NE
NE
300
NE
NE
30
NE
30
100
30
NE
NE
NE
150.2 (137.9, 9162.5)
NE
NE
463.5 (431.9, 495.1)
NE
187.6 (172.7, 202.5)
170.8 (155.9, 185.7)
75.4 (70.1, 80.7)
NE
NE
NE
300, 1000
NE
NE
30, 100, 300, 1000
NE
30, 100, 300, 1000
100, 300, 1000
30, 100, 300, 1000
NE
NE
NE
−2.7 (−3.0, −2.4)
NE
NE
4.9 (4.5, 5.3)
NE
4.4 (3.7, 5.1)
−1.1 (−1.1, −1.1)
−1.1 (−1.2, −1.0)
Liver
er1
vtg1
vtg2
er2˛
300
300
300
300
NE
NE
NE
NE
300, 1000
300, 1000
300, 1000
300, 1000
−3.4 (−3.9, −3.0)
−29.7 (−32.6, −26.8)
−20.8 (−23.4, −18.2)
1.8 (1.6, 2.0)
NE
1000
NE
300
NE
NE
NE
NE
NE
1000
NE
300
NE
−4.9 (−5.6, −4.2)
NE
0.9 (0.9, 1.0)
Females
Males
Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
LOEC: the lowest observed effect concentration, ␮g/L; EC50 : the concentration to induce half of the maximal response, ␮g/L; OMRV: observed maximal response value, fold change relative control; OECPs: observed effect
concentration points, ␮g/L; NE: not estimated because the number of genes with statistically significant response was ≤1 or the maximal response was not reached.
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Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
previous studies suggested that CYP19A activity was a more sensitive target of prochloraz than other steroidogenic CYPs (e.g., CYP17)
and plasma volume was limited and could only be used for detection of a single hormone (Ankley et al., 2009). Therefore, only E2
was quantified in plasma. Consequently, we expected exposure
to prochloraz to impair the synthesis, decreasing concentration of
E2 in blood plasma. Results observed in this study are consistent
with this expectation, with a significant decrease in concentrations
of E2 in blood plasma in females and males exposed to 300 or
1000 ␮g/L prochloraz. In fish, synthesis of vtg in the liver is under
the control of er, and is induced by estrogens, such as E2. Therefore, decreased concentrations of E2 in blood plasma would result
in down-regulated expression of er1 and vtgs in liver. The results
observed in this study were consistent with these hypotheses. Previous studies also reported that treatment with 300 or 1000 ␮g/L
prochloraz significantly decreased concentrations of E2 in blood
plasma and down-regulated the expression of er1 and vtgs in liver
of small fish, including zebrafish, medaka and fathead minnow
(Ankley et al., 2005, 2009; Liu et al., 2011; Skolness et al., 2011;
Zhang et al., 2008a).
Results of this study were consistent with those of previous
observations that small fish (e.g., fathead minnow, medaka and
zebrafish) have the capacity to mount a compensatory response to
the direct inhibitory effects of prochloraz on blood plasma E2 concentration by up-regulating expression of genes included in brain
and gonad (Ankley et al., 2009; Liu et al., 2011; Zhang et al., 2008a).
Similar to previous studies, where time- or dose-dependent upregulation of genes included in brain and gonad were observed in
small fish exposed to prochloraz (Ankley et al., 2009; Liu et al., 2011;
Zhang et al., 2008a), the expression of some genes was also induced
in the present study. However, in this study, we provided more
details and insights on dose-dependent compensatory response
along HPG-axis and liver by using more exposure doses compared
with previous studies.
In female and male zebrafish exposed to low and medium concentration prochloraz, compensatory responses occurred in brain
and both brain and gonad, respectively in this study. In fish, gnrh
can induce the synthesis of fsh and lh by gnrhr in brain, and then
fsh and lh are secreted by the pituitary and bind to their receptors (fshr and lhr) in the gonad to induce steroidogenesis (Liu et al.,
2011; Zhang et al., 2008a). In addition, sex hormones (e.g., E2)
in blood can enter into brain and work by their receptors (e.g.,
er) for negative feedback regulation (Liu et al., 2011; Zhang et al.,
2008a). In this study, up-regulation of gnrhrs, lhˇ and ers in brain
was observed after exposure to low and medium concentration
prochloraz for 4 days. Direct effects of prochloraz on the expression of these genes were excluded in previous study using in vitro
brain explant exposure (Liu et al., 2011), therefore, up-regulation
of these genes was considered as a compensation response for
decreased concentration of E2. However, in this study, exposure
to low concentrations prochloraz did not decrease concentrations
of E2 in blood plasma and vtgs expression in liver. A possible explanation was that the inhibitory effect of prochloraz on E2 might be
transitory and occurred in initial stage of exposure, but concentration of E2 recovered due to compensation responses and returned
to control levels by the end of 4-day exposure. This has been confirmed by designed experiments in fathead minnows (Ankley et al.,
2009; Villeneuve et al., 2009), where the inhibitory effects of low
concentration prochloraz or fadrozole on concentrations of E2 in
blood plasma in fathead minnow only occurred within 1 day of
exposure but returned to the control level by the end of the 8day exposure phase. In previous studied using fathead minnow
or Japanese medaka medaka (Oryzias latipes) as animal models,
expression of gnrhrs, lhˇ and ers kept unchanged after exposure
to prochloraz (Ankley et al., 2009; Skolness et al., 2011; Zhang
et al., 2008a), although expression of chicken-II-type gnrh (cgnrh
II) was significantly up-regulated in female medaka after 3 ␮g/L
prochloraz exposure for 7 days (Zhang et al., 2008a). However, consistent with the results of this study, our previous study found that
exposure to 300 ␮g/L prochloraz for 12 or 48 h caused a strong upregulation of several gnrhrs in brain of female zebrafish (Liu et al.,
2011). Therefore, these differences might be explained by species
differences. For low concentration exposure, our results demonstrated that compensatory up-regulation of gene expression only
occurred in brain, not in gonad and liver, but the compensation
is successful since no significant effect on concentrations of E2
were observed by the end of the 4-day exposure. These results
suggested that other feedback mechanisms might be included. For
example, in a previous study it was reported that treatment with
LH in immature rat leydig cells significantly increased the activity
of steroidogenic enzyme (CYP11A) (van Haren et al., 1995). Therefore, the feedback mechanisms might have also included responses
at other levels than transcription, such as increased enzyme activity or protein synthesis. Further studies are needed to explore the
details of other compensation responses. For medium concentration exposure, compensatory responses included up-regulation of
steroidogenic genes except for genes included in brain above. Similar compensatory up-regulations of these genes were also reported
in fathead minnow and medaka exposed to 30-␮g/L prochloraz for
8 days (Ankley et al., 2009; Liu et al., 2011). Similarly, exposure
to 30 or 100 ␮g/L prochlorza did not change plasma E2 concentrations and vtgs expression of liver in female and male zebrafish
in the present study, suggesting the effectiveness of compensation
responses.
Transcriptional compensation seems to involve up-regulation
of genes in brain, gonad and liver in female and male zebrafish
after exposure to greatest concentration prochloraz. In this study,
up-regulation of gnrhrs, lhˇ and ers was also observed in fish
exposed to the greatest concentrations, which suggests consistency
in responses. In gonad, abundances of several genes were increased
and cyp17 and cyp19a was the most up-regulated gene. In a previous study the most up-regulated gene in female fathead minnow
exposed to 300-␮g/L prochloraz codes was cyp19a (Ankley et al.,
2009). In medaka, a significant up-regulation of cyp17 and cyp19a
was also observed in females exposed to 300-␮g/L prochloraz for
7 days (Zhang et al., 2008a). Consistent with the results of the
present study and previous published papers (Ankley et al., 2009;
Liu et al., 2011; Zhang et al., 2008a), a study reported that compensatory responses of female fathead minnow to cyp19a inhibitor
(fadrozole)-induced decrease in concentrations of E2 in blood
plasma were associated with up-regulation of steroidogenic genes
in ovaries (Villeneuve et al., 2009). In liver, besides down-regulation
of er1 and vtgs observed, exposure to the greatest concentration
prochloraz up-regulated expression of er2˛. Down-regulation of
vtgs and er1 was considered to be a direct effect of prochlorazinduced decreases in production of E2, and up-regulation of er2˛
might be a mechanism of compensation to decreased production
of vtgs. In a recent study it was found that er2 was also involved
in E2-induced vtg synthesis in fish (Yost et al., 2014). Therefore,
further study is needed to explore the possible roles of er2␣ in fish
exposed to prochloraz in future.
In summary, in this study, dose-dependent responsive behaviors of genes involved in HPG axis and liver were investigated
and linkages between compensatory responses occurred in brain,
gonad and liver in terms of degrees of prochloraz stresses. Two
points are especially notable in this regard. First, in low-dose
exposure groups transcriptional compensation responses of HPG
axis occurred only in brain, suggesting that other down-stream
compensatory mechanisms might be involved, therefore, further
studies are needed to explore those possibilities. Second, upregulation of er2˛ in liver was considered as a compensatory
mechanism for decreased vtgs production due to exposure to
Y. Dang et al. / Aquatic Toxicology 160 (2015) 69–75
prochloraz. This result suggests that compensatory responses also
occur in liver. However, these responses, although critical to understanding the mechanisms of chemicals, need to be examined and
integrated in a broader system to support more reliable predictions of toxicity of chemicals.[1] Additionally, it should be noted
that in low and medium concentration groups, E2 concentrations
kept unchanged and we speculated that transcriptionally compensatory responses (e.g., up-regulation of genes involved in HPG axis
and liver) were successful in maintaining homeostasis. However,
we did not measure protein contents or activities of enzymes to further support our hypothesis. Therefore, further studies are needed
in future to explore these possibilities.
Acknowledgements
This work was supported by Huazhong Agricultural University
Scientific & Technological Self-innovation Foundation (Program
No. 2014RC001) to Dr. Chunsheng Liu. This work was also supported by the National Natural Science Foundation of China
(31370525) and the Fundamental Research Funds for the Central
Universities (2014PY027) to Dr. Jianghua Wang. Prof. Giesy was
supported by the program of 2012 “High Level Foreign Experts”
(#GDW20123200120) funded by the State Administration of Foreign Experts Affairs, the P.R. China to Nanjing University and the
Einstein Professor Program of the Chinese Academy of Sciences.
He was also supported by the Canada Research Chair program, a
Visiting Distinguished Professorship in the Department of Biology
and Chemistry and State Key Laboratory in Marine Pollution, City
University of Hong Kong.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.
2015.01.003.
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